Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype.

This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied.

Methods: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity.

Results: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival.

Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively.

The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential.

Conclusions: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.

Background
Patients with rhabdomyosarcoma (RMS) who complete therapy typically undergo 4 years of surveillance imaging despite lack of evidence that this improves outcomes. We compared overall survival (OS) between patients in whom progression or relapse was detected by routine clinical evaluation or by imaging.

Procedure
Children with progressive or relapsed RMS treated at Texas Children's Hospital between 1992 and 2012 were identified and their records were reviewed. Survival time after progression or relapse was compared between two groups: (1) patients in whom progression or relapse was suspected on the basis of clinical history, symptoms, laboratory evaluation, or physical exam; and (2) patients whose progression or relapse was initially detected by imaging.

Results
Of the 43 children with progressive or relapsed RMS, 26 (60%) had metastatic disease at diagnosis and 19 (44%) had alveolar histology. With a median follow up time of 5 years in six survivors, there was no difference in OS between patients in whom progression or relapse was diagnosed based on imaging (n = 15) or by clinical evaluation (n = 28) (3-year OS 20% vs. 11%, respectively, P = 0.38). Disease extent, primary site, and risk group at diagnosis were associated with survival after progression or relapse.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/coa.12527

AbstractObjectives
To determine the hearing status of survivors treated for head and neck rhabdomyosarcoma (HNRMS) at long-term follow-up.

Design
Cross-sectional long-term follow-up study.

Setting
Tertiary comprehensive cancer center.

Participants
Survivors treated for head and neck rhabdomyosarcoma during childhood in two concurrent cohorts; survivors in London had been treated with external beam radiotherapy (EBRT-based local therapy); survivors in Amsterdam were treated with AMORE (Ablative surgery, MOld technique afterloading brachytherapy and surgical REconstruction) if feasible, otherwise EBRT (AMORE-based local therapy).

Methods
We assessed the hearing thresholds, the number of patients with clinically relevant hearing loss, and hearing impairment graded according to the Common Terminology Criteria for Adverse Events version 4.0 (CTCAEv4) and Boston criteria. Furthermore, we compared hearing loss between survivors treated with EBRT-based local therapy (London) and AMORE-based local therapy(Amsterdam).

Results
Seventy-three survivors were included (median follow-up 11 years). We found clinically relevant hearing loss at speech frequencies in 19% of survivors. Multivariable analysis showed that survivors treated with EBRT-based treatment and those with parameningeal tumors had significantly more hearing impairment, compared to survivors treated with AMORE-based treatment and non-parameningeal tumors.

Conclusions
One in five survivors of HNRMS developed clinically relevant hearing loss. AMORE-based treatment resulted in less hearing loss compared to EBRT-based treatment. As hearing loss was highly prevalent and also occurred in survivors with orbital primaries, we recommend systematic audiological follow-up in all HNRMS survivors.

Background:
Rhabdomyosarcoma (RMS) in a pediatric tumor of myogenic origin. It includes two subtypes: embryonal and alveolar. Embryonal RMS (ERMS) cells express key myogenic factors such as MyoD and Myogenin, but proliferate indefinitely and have lost the ability to terminally differentiate into skeletal myofibers. Differently from the alveolar tumors bearing specific chromosomal translocations, ERMS has cytogenetic aberrations and molecular deregulations of pathways regulating senescence, proliferation and differentiation. It has been shown that SKP2, an F-box protein and a component of the ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box), is over-expressed in RMS primary samples and correlates with a dismal outcome. Therefore, we sought here to investigate the regulation of SKP2 and its role in ERMS.

Methods:
We modulated SKP2 expression through silencing, by using a siRNA validated in the literature, and forcing its expression through retroviral infection. In parallel, we investigate the effect of Notch signaling modulation on SKP2 expression.

Results:
SKP2 silencing resulted in cell cycle slowdown in both normal myoblasts and ERMS cells. Down-regulation of Notch1 led to SKP2 reduction while that of Notch3 supported SKP2 expression. Cosilencing SKP2 and Notch3 gave raise to myoblast-like structure formation in ERMS and facilitated myoblasts fusion. Finally, using a SKP2 inhibitor ERMS cell proliferation was completely blocked.

Conclusion:
Altogether, these preliminary experiments suggest that SKP2 could be regulated by Notch signaling in ERMS and that its inhibition hampers tumor cell proliferative capability.

Myogenic regulatory factors and their role in embryonal rhabdomyosarcoma

Abstract
Rhabdomyosarcoma (RMS) is a pediatric sarcoma of muscle. RAS pathway activation is the dominant oncogenic driver event in fusion negative RMS, which includes the Embryonal RMS (ERMS). We have previously shown in a transgenic zebrafish model of kRASG12D induced ERMS that tumor-propagating potential is confined to molecularly defined cells that express Myf5, m-Cadherin, c-Met and additional satellite cell markers. MYF5 and MYOD1 are bHLH myogenic regulatory factors (MRFs) that orchestrate skeletal muscle differentiation during development and regeneration. Both are sufficient to reprogram human mesenchymal cells into a myogenic fate. Importantly, these same factors are highly expressed a subset of mouse and human ERMS. Given that Myf5 is highly and specifically expressed in the TPC compartment in our ERMS model, we hypothesized that Myf5 and its transcriptional targets may regulate self-renewal and growth of TPCs.

To address this question, we utilized transgenic zebrafish and expressed Myf5 in differentiated ERMS cells that lack proliferative capacity and can not make tumors when transplanted into recipient fish. Induced expression of Myf5 was sufficient to confer tumor-propagating ability to differentiated populations of ERMS cells. These “Induced TPCs” proliferate and generate aggressive tumors that re-express satellite cell markers and yet retain expression of mature muscle markers. Next, we assessed if Myf5 is required for ERMS initiation in the zebrafish model. Remarkably, kRASSG12D-induced ERMS could be generated in Myf5 loss-of-function zebrafish. Moreover, Myf5-deficient ERMS could regrow following transplantation into rag2E450fs immune-compromised zebrafish; however, these tumors are histologically and molecularly distinct when compared with ERMS arising in wild-type fish. Given the redundancy of function between Myf5 and MyoD in muscle and their differential expression in satellite cells and muscle progenitors, our current hypothesis is that Myf5-deficient zebrafish ERMS likely recapitulate a molecularly-distinct class of human ERMS. This idea is consistent with the fact that MYF5 is expressed in only 50% of primary human ERMS and a small fraction of human cell lines. Importantly, knock down experiments in human ERMS cell lines confirm independent roles of either MYF5 or MYOD1 in maintenance of ERMS cell viability in vitro.

Collectively, our results support a previously unappreciated role for bHLH MRFs in ERMS cell survival and intra-tumor functional heterogeneity and suggest that myogenic factor expression may define unique subtypes of ERMS.

Abstract
Background: Rhabdomyosarcoma (RMS) is most common soft tissue sarcoma in childhood and adolescence. There are two major subtypes of RMS - alveolar RMS (ARMS) and embryonal RMS (ERMS). With current treatment modalities the 5 year survival rate of patients with metastatic disease is only about 30%, thus necessitating development of novel targeted therapeutic strategies. Fibroblast Growth Factor Receptor 4 (FGFR4) is highly differentially expressed gene and activating mutations in FGFR4 is associated with increase in RMS metastasis. We hypothesize that FGFR4 can be exploited as a potential therapeutic target in RMS. Monoclonal antibodies (mAbs) against specific cancer cell surface antigens, have gained importance as therapeutic agents in other cancer types. Therefore, mAbs against FGFR4 may be developed either alone or conjugated with a chemotherapeutic agent for treatment of RMS.

Methods: We have developed 15 mAbs that are of mouse, rabbit and human origin. The immunogen for rabbit antibodies is human FGFR4 extracellular domain (ECD), while human FGFR4-Fc chimeric protein or hFGFR4 transfected cells were used for mouse antibodies and were produced using hybridoma technology. Using recombinant DNA technology, human immunoglobulin library was selected using FGFR4 ECD to derive human anti-FGFR4 mAbs. All mAbs were purified using affinity chromatography and reactivity was confirmed by ELISA. These mAbs were tested for their ability to bind cell surface FGFR4 and internalization of mAb bound FGFR4 (upon incubation at 37°C) by flow cytometry using RMS cell lines. To further characterize these antibodies we have performed immunohistochemistry (IHC) on tissue microarrays (TMA) of normal tissue, xenografts of RMS cell lines, and primary tumor from RMS patients. Finally, to select a potential mAb candidate as therapeutic agent, we tested appropriate secondary antibody-drug conjugate (ADC) using cell based cytotoxic assay.

Results: We observed that tested mAbs bind to both ARMS and ERMS cell lines. The level of FGFR4 expression is variable in both subtypes. Internalization assays demonstrated mAbs bound FGFR4 was internalized in RMS cell lines. Analysis by IHC on TMAs suggests that FGFR4 protein is expressed at a considerably higher level in RMS tumor tissue and xenografts compared to normal tissue. Based on these findings, we are currently screening the mAbs for their ability to elicit a cytotoxic response either alone or in conjunction with secondary ADC.

Conclusion: These results suggest anti-FGFR4 mAbs can be used as therapeutic intervention for RMS. High expression of FGFR4 in other cancers such as prostate, melanoma, lung, breast, colorectal, and gastric cancers suggests the potential use of anti-FGFR4 mAbs and their derivatives in these cancers.

Abstract
Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric muscle cancer with extremely poor prognosis at relapse. Work from our group has identified the tumor-propagating cell (TPC) in a transgenic zebrafish model of kRASG12D-induced ERMS that is responsible for driving continued tumor growth and relapse. The TPC is molecularly similar to an activated muscle satellite-cell and expresses myf5, c-met, and m-cadherin. Building on these observations, we have identified the Notch pathway as a potent enhancer of ERMS self-renewal and TPC number. Specifically, bulk tumor limiting dilution cell transplantation experiments revealed that TPCs are increased 10-fold in kRASG12D expressing ERMS that co-express activated intracellular Notch1 (ICN1). This increase in TPC number is partly the result of ICN1 expressing ERMS exhibiting a 3-fold expansion of relapse-driving myf5-GFP+/mylz2-mCherry-negative ERMS-cell population. Unexpectedly, cell transplantation experiments revealed that Notch pathway activation also conferred tumor-propagating ability to the myf5-GFP+/mylz2-mCherry+ mid-differentiated ERMS cells - a population of cells previously shown to lack self-renewal capacity. Single cell engraftment studies uncovered that NOTCH activation caused cells to oscillate between the TPC and mid-differentiated ERMS molecular states that was not observed in kRASG12D-expressing ERMS, suggesting that Notch has important roles in both self-renewal and cell state transitions.

Next, we validated our findings in human ERMS where NOTCH1 is highly expressed both in tumors and ERMS-cell lines. We show important roles for NOTCH in regulating self-renewal and differentiation in human ERMS. Specifically, human ERMS cells that expressed activated NOTCH1 had elevated sphere-colony formation, a surrogate for self-renewal in vitro. By contrast, shRNA knockdown of NOTCH1 resulted in decreased sphere-colony formation and robust terminal differentiation of ERMS cells into late-stage, myosin-expressing myoblasts. Moreover, we identified that NOTCH1 directly activated SNAI1 expression and was required for both efficient sphere formation and differentiation in ERMS cell lines. SNAI1 is commonly over-expressed in human ERMS and its expression is positively correlated with NOTCH1. Taken together, our data indicate that Notch signaling is an important modifier of human ERMS acting to regulate both TPC self-renewal and differentiation. Notch and/or SNAl1 pathway inhibition may have potential benefit for a subset of patients with relapsed ERMS.

Furin activity: A driver of rhabdomyosarcoma progression

Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis and survival rate is often very poor and therefore new therapy approaches are required. Thus, we focus on 1) employing RMS-specific peptides for targeted delivery of chemotherapeutics to the tumor site and 2) identifying novel therapeutic targets. In a previous study we discovered that RMS-homing peptides bind preferably to the proprotein convertase furin. Initial investigations confirmed a high expression of furin throughout different pediatric soft tissue sarcoma types and hinted that furin promotes the tumorigenic phenotype of RMS cells in vitro as well as in corresponding xenografts in vivo. Here we present a novel approach of tetracycline-induced shRNA-based silencing of furin in vitro and in vivo in order to investigate in depth the role of furin in RMS progression. Furin depletion was confirmed at mRNA, protein and activity level and led to impaired maturation furin substrates insulin like growth factor 1 receptor (IGF1R) and transforming growth factor beta 1 (TGFβ-1). We found that loss of furin activity suppresses the malignant phenotype of Rh30 cells by decreasing proliferation and affecting formation of colonies. Furthermore, we observed increased caspase 3/7 activity and enrichment of nucleosomes in the cytoplasm upon furin depletion, thus hinting initiation of apoptotic processes. Induction of furin silencing in RMS xenografts in NOD/SCID mice delayed tumour growth, indicating a crucial role of furin in early phases of tumour growth. Taken together, our data underscore the importance of furin for RMS progression and therefore targeting the activity of furin represents a promising tool for treatment of RMS.

]]>http://focusonrhabdo.org/furin-activity-a-driver-of-rhabdomyosarcoma-progression/feed/01154PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesishttp://focusonrhabdo.org/pax3-foxo1-is-essential-for-initiation-but-not-for-recurrence-during-rhabdomyosarcoma-tumorigenesis/
http://focusonrhabdo.org/pax3-foxo1-is-essential-for-initiation-but-not-for-recurrence-during-rhabdomyosarcoma-tumorigenesis/#respondTue, 18 Aug 2015 11:20:29 +0000http://focusonrhabdo.org/?p=1152

PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis

Abstract
The PAX3-FOXO1 fusion gene, which is generated by a 2; 13 chromosomal translocation, is a characteristic feature of fusion-positive rhabdomyosarcoma (RMS), a major RMS subtype associated with aggressive behavior and poor prognosis. This study utilizes a novel inducible expression system in human myoblasts to dissect the molecular mechanism and contribution of PAX3-FOXO1 in RMS tumorigenesis.A human myoblast cell line (immortalized by BMI1 and TERT expression) was transduced with a retroviral construct that constitutively expresses MYCN and a lentiviral based- doxycycline inducible construct that variably and reversibly expresses PAX3-FOXO1. Focus formation and animal xenograft experiments were performed to study transformation in vitro and tumorigenesis in vivo, respectively. Myogenic differentiation was assessed by light microscopy and by western blot or immunohistochemical assays of muscle-specific protein expression.PAX3-FOXO1-transduced myoblasts treated with doxycycline demonstrated a time- and dose-dependent increase in expression of PAX3-FOXO1 mRNA and protein and its downstream targets genes; doxycycline withdrawal led to cessation of fusion protein expression. Though myoblasts expressing PAX3-FOXO1 or MYCN alone did not show any evidence of transformation in culture, combined PAX3-FOXO1 and MYCN expression resulted in myoblast transformation. Under differentiation-promoting culture conditions, combined PAX3-FOXO1 and MYCN expression inhibited myogenic differentiation. Intramuscular injection of myoblasts with MYCN and PAX3-FOXO1 resulted in rapid RMS tumor formation in NOD-SCID mice when fusion protein expression was induced by feeding mice a doxycycline-containing diet. Myoblasts with MYCN expression alone did not form any tumors while PAX3-FOXO1 induction without MYCN expression resulted in RMS tumors only after a much longer latency period. After tumors formed from myoblasts expressing PAX3-FOXO1 with or without MYCN, down-regulation of PAX3-FOXO1 expression by doxycycline withdrawal resulted in tumor regression associated with widespread myogenic differentiation. However, the regressed tumors slowly grew back in the absence of doxycycline induction demonstrating a PAX3-FOXO1-independent oncogenic mechanism for recurrence.The PAX3-FOXO1 fusion protein collaborated with MYCN in the initial stage of RMS tumorigenesis to promote dysregulated cell proliferation and inhibit myogenic differentiation. Though most cells in the initial tumor were dependent on the fusion protein, recurrent tumors formed in which the fusion protein was not required to maintain the tumorigenic phenotype.

The role of childhood immunizations and infections on the risk of pediatric rhabdomyosarcoma

Background: Rhabdomyosarcoma (RMS) is a rare, highly malignant childhood cancer with an annual incidence of four cases per million and approximately 350 new cases are diagnosed in the U.S. annually. Due to its rarity, relatively little is known about the etiology of this childhood malignancy. Early immunologic challenges such as immunizations and infections have been hypothesized to be protective in certain malignancies. To our knowledge, these associations have not been studied in relation to childhood RMS risk. The purpose of our study was to better characterize the association between childhood immunizations and infections and RMS risk using data from the largest case-control study of RMS to date.

Materials and Methods: Cases (n = 322) were previously enrolled in the Intergroup Rhabdomyosarcoma Study, which became a part of the Children's Oncology Group in 2000. Controls (n = 322) were selected by random-digit telephone dialing and pair-matched to cases on race, sex, and age. We evaluated RMS risk based on whether parents reported that their child was up-to-date on immunizations and had completed their DPT (diphtheria, pertussis and tetanus), oral polio, and MMR (measles, mumps and rubella) series. In addition, we evaluated whether the child ever had previous infections including mumps, pneumonia, rubella, rubeola, pertussis, tuberculosis, meningitis, scarlet fever, and chickenpox. Conditional logistic regression models were used to calculate an adjusted odds ratio (aOR) and 95% confidence interval (CI) for each exposure, adjusted for age, race, and sex.

Results: Having up-to-date immunizations had a protective effect on childhood RMS (aOR = 0.22, 95% CI: 0.11-0.44). More specifically, children who did not receive the complete vaccination series of DPT (aOR = 1.70 95% CI: 1.18-2.50) or did not receive the MMR vaccine (aOR = 1.59, 95% CI: 1.04-2.44) had an increased risk of being diagnosed with childhood RMS. Most infectious conditions examined were negatively associated with childhood RMS; however, these findings were not statistically significant (p>0.05).

Conclusions: This is the first study of its kind to provide evidence that receiving childhood immunizations may be protective against RMS risk. Although the effect estimates suggested a possible protective role of childhood infections against RMS risk, these findings were not statistically significant. Future studies will be needed to further evaluate the role of immunizations and childhood infections in the development of this tumor.